Genome-wide analysis of human DNA in small cell populations is becoming increasingly important in modern medicine in applications ranging from cancer to understanding tissue development. While there are many commercially available macroscale sample preparation kits and microfluidic devices for nucleic acid isolation, it remains extremely challenging to efficiently extract and purify DNA from a few cells, let alone a single cell. The traditional vial-based extraction techniques utilize electrostatic interactions of DNA with biochemically functionalized magnetic microparticles or spin columns that are used to separate nucleic acids from the rest of the cell lysate. An appreciable fraction of genomic DNA is always lost during the purification process, which presents a serious problem when the whole genome of small cell populations such as rare cancer cells or stem cells needs to be analyzed. Additional losses are introduced during sample manipulation when the purified DNA is eluted from the microparticles or spin columns. The utility of DNA extraction tools for medically relevant genome-wide studies would be enhanced by integrating these tools with single-molecule spectroscopy, imaging, and sorting systems (Refs. A1-A3).
The macroscale DNA extraction techniques have been recently investigated by various research groups (Refs. A4-A13) and implemented in microfluidic systems that provide handling and manipulation of small sample and reagent volumes in engineered microstructures. Microfluidic devices could perform the analysis automatically in an enclosed system which reduces the chance for human error and cross contamination. These devices may also reduce the time and the cost of the analysis by taking advantage of high reaction rates at the microscale and generally provide higher extraction efficiencies by utilizing features with high surface-to-volume ratios for improved DNA extraction, but they still rely on DNA adsorption to silica or other biochemically functionalized surfaces. The binding affinity is extremely sensitive to temperature, pH, and buffer composition which need to be optimized carefully to minimize DNA losses. Even after meticulous optimization it is difficult to ensure that all the DNA fragments get adsorbed and the whole genome is represented in purified extracts obtained from a few cells. Fundamentally different approaches to genomic DNA capture should therefore be explored to improve and facilitate the extraction efficiency.
There are a variety of commercially available DNA extraction kits that advertise the ability to extract DNA from a few cells. See e.g. Applied Biosystems® Arcturus® PicoPure® DNA. However, unlike microfluidic devices, these kits do not perform purification of the extracted DNA and simply focus on creating buffer chemistries that do not interfere with PCR amplification. Additionally, whole genome amplification has amplification induced errors and template biases that are a problem with such systems. There is a need for DNA purification systems for single-molecule fluorescence studies not provided in the art. There is also a need for systems that decrease the amplification induced errors for applications such as whole genome amplification.
The present invention is directed to overcoming these and other deficiencies in the art.